Abstract/Summary Immunologic memory is a cardinal feature of the adaptive immune system critical for lifelong immunity to pathogens and, hence, survival of the human species. Memory cells differentiate from nave precursors by paths that are not entirely clear and acquire phenotypes to allow them to carry out specific functions to provide lifelong immunity. It is generally believed that acquisition of these new phenotypes result from alterations in the transcriptional program by epigenetic modifications at specific target gene loci that enable memory cells to express genes critical for their function. It is easy to see how understanding these responses is important as it could contribute to better vaccine design, enhancement of memory responses to fight against infection or cancers, or faulty regulation of memory responses may contribute to autoimmune diseases, and thus positive or negative regulation of memory responses could have significant health benefits. Activation and repression of transcription of protein-coding genes is key to all facets of biology. Recently the field has learned that much of the genome is transcribed. Two new classes of RNA regulatory elements include long non-coding RNAs (lncRNA) and RNAs transcribed from enhancers (eRNAs). Determining biologic roles of these new classes of RNAs is an active area of investigation still in its infancy. A recent review addressed this question: ?Functional roles of enhancer transcription in gene regulation. Three non-exclusive models may underlie the functions of enhancer transcription: the transcription process and enhancer RNAs (eRNAs) are non-functional and are merely transcriptional noise (part A); the act of enhancer transcription mediates function (part B); and genes on the same chromatin fiber (cis), or potentially on other chromosomes (trans), are regulated by an eRNA (part C).? Our central hypothesis is that these RNAs are functional and we propose to address their functions in CD4+ effector memory cells (TEM). We provide preliminary data to support this premise. A corollary is that one mechanism of action of these enhancer-associated RNAs is to bind transcription factors (TFs) and tether them to chromatin. YY1 is one example of a TF that may act in this manner and we provide preliminary data that NF-kB is a second, also supporting our premise. We propose successful completion of studies described herein will not only improve our understanding of this class of RNA molecules but will also improve our understanding of how immunologic memory is maintained. Large numbers of discrete lncRNAs are transcribed from gene loci critical for maintenance of TEM transcriptional programs and are localized at enhancers. The following specific aims are proposed: (1) To determine if IFNG-locus specific enhancer-associated lncRNAs are required for IFNG-AS1, IFNG, IL26, and/or IL22 expression by TEM cells and define epigenetic underpinnings, and (2) To identify biologic functions of additional TEM enhancer associated RNAs and determine if TF binding is a common property.